Binding molecules for human factor VIII and factor VIII-like proteins

ABSTRACT

It is an object of the present invention to provide novel binding molecules for factor VIII and factor VIII-like proteins. Preferred binding molecules of the present invention exhibit not only distinct characteristics for binding of the target factor VIII polypeptides but also specific and desirable characteristics for release (elution) of the target polypeptides. Especially preferred binding molecules according to the invention are short polypeptide sequences, characterized by a stable loop structure.

RELATED APPLICATIONS

This Application is a divisional of U.S. Ser. No. 10/272,497, filed Oct.15, 2002, now U.S. Pat. No. 7,112,438 which is a continuation-in-part ofU.S. Ser. No. 09/756,594, filed Jan. 8, 2001, now U.S. Pat. No.6,492,105, which is a divisional of U.S. Ser. No. 09/224,785, filed Jan.4, 1999, U.S. Pat. No. 6,197,526, the contents of which are incorporatedherein by reference.

BACKGROUND

Classical hemophilia A is the result of a chromosome X-linked deficiencyof blood plasma coagulation factor VIII and affects almost exclusivelymales with a frequency of about 1 case per 10,000. The X-chromosomedefect is transmitted by female carriers who do not themselves have thedisease. Factor VIII is also known as antihemophilic factor (AHF),hemophilic factor A, platelet cofactor, thromboplastinogen,thrombocytolysin, and antihemophilic globulin (AHG). The designation“factor VIII:C” is used to indicate that it is the compound that affectsclotting. Factor VIII is a high molecular weight protein of 280 kDa andis composed of two polypeptide chains of 200 kDa and 80 kDa,respectively. Andersson et al., Proc. Natl. Acad. Sci. U.S.A.,83:2979-2973 (1986). These chains are held together by a metal ionbridge.

The principal symptom of hemophilia A is bleeding without clotting orcoagulation. Prior to the discovery that administration of factor VIIIconcentrates could ease the symptoms of an individual diagnosed with thedisease, the average life expectancy of a sufferer was about 20 years.

Until recent years, the major source of factor VIII for therapeuticpurposes was normal blood plasma; however factor VIII isolated by thismethod, while of some use, has several important disadvantages. Forinstance, factor VII isolated from blood plasma is fairly impure,typically having a specific activity of less than 2 units factor VIII/mgprotein and an overall factor VIII content of less than 1%.Additionally, the purification process is expensive because the startingmaterial, i.e., human plasma, is expensive. Many precautions must alsobe taken to decrease the risk of transmitting infectious agents to thepatient. For example, human immunodeficiency virus (HIV), Hepatitis Bvirus, Hepatitis C virus and other disease-causing agents are commonlydetected in donated blood. Another disadvantage of using factor VIIIobtained by this method is that approximately one-tenth of the patientswith severe hemophilia A develop antibodies against factor VIII, makingthe disease difficult to treat.

Research efforts have focused on the development of methods for creatingand isolating highly purified, biologically active factor VIII infull-length and derivative forms. Advantages of a highly purifiedprotein include reduced levels of extraneous proteins in the therapeuticmix as well as a decreased likelihood of the presence of infectiousagents. A more purified form of factor VIII can also be administered insmaller doses, possibly reducing the risk of developing anti-factor VIIIantibodies, as lower doses would be less challenging to the immunesystem.

Significant steps have been taken toward the recombinant production offactor VIII beginning with the isolation of biologically active factorVIII fragments. See, Andersson et al., U.S. Pat. No. 4,749,780;Andersson et al., U.S. Pat. No. 4,877,614. The gene encoding thefull-length human factor VIII protein was isolated by taking advantageof its sequence homology with porcine factor VIII. See, Toole et al.,U.S. Pat. No. 4,757,006. Toole et al. also report the expression ofhuman and porcine protein having factor VIII:C procoagulant activity.

However, severe side effects involving the production of anti-factorVIII antibodies still exist with the administration of the proteinisolated from both human and non-human sources. Antibodies that reactwith human factor VIII:C are also known to react, to a certain extent,with factor VIII:C from other species, and porcine factor VIII itself isantigenic in humans. Also, non-hemophiliacs can develop or acquire thedisease when their immune systems become sensitized to factor VIII:C.

As a possible solution to this problem, a truncated, lower molecularweight protein exhibiting procoagulant activity has been designed. See,Toole, U.S. Pat. No. 4,868,112. Toole reported an alternative method oftreatment with lower molecular weight porcine factor VIII ofapproximately 2000 amino acids exhibiting similar procoagulant activityas full-length factor VIII. Evidently, the removal of certain aminoacids and up to 19 of the 25 possible glycosylation sites, reduced theantigenicity of the protein and thereby the likelihood of developinganti-factor VIII antibodies. However, one difficulty with thedevelopment of recombinant factor VIII is achieving production levels insufficiently high yields.

Recently, deleted factor VIII cDNA molecules coding for recombinantfactor VIII derivatives, which were likely to give sufficiently highyields of a biologically active recombinant factor VIII protein for usein an industrial process for a pharmaceutical preparation have beendeveloped. See, Almstedt et al., U.S. Pat. No. 5,661,008. Almstedt etal. designed a modified factor VIII derived from a full-length factorVIII cDNA, that, when expressed in animal cells, produced high levels ofa factor VIII-like protein with factor VIII activity. The proteinconsisted essentially of two polypeptide chains derived from humanfactor VIII, the chains having molecular weights of 90 kDa and 80 kDa,respectively.

According to the Almstedt et al. process, the factor VIII cDNAs areassembled into transcription units and introduced into a suitable hostsystem for expression. The cell lines can be grown on a large scale insuspension culture or on solid support. The protein produced in theculture medium is then concentrated and purified. The final activeprotein is made up of amino acids 1 to 743 and 1638 through 2332 ofhuman factor VIII This polypeptide sequence is commercially known asrFVIII-SQ or REFACTO®. See also, Lind et al., Euro. J. Biochem.,232:19-27 (1995). Other forms of truncated FVIII can also be constructedin which the B-domain is generally deleted. In such embodiments, theamino acids of the heavy chain, consisting essentially of amino acids 1through 740 of human Factor VIII and having a molecular weight ofapproximately 90 kD are connected to the amino acids of the light chain,consisting essentially of amino acids 1649 to 2332 of human Factor VIIIand having a molecular weight of approximately 80 kD. The heavy andlight chains can be connected by a linker peptide of from 2 to 15 aminoacids, for example a linker comprising lysine or arginine residues, oralternatively, linked by a metal ion bond.

Currently, there is a need in the field for efficient and cost-effectivemethods for obtaining purified, active factor VIII directly from varioussolutions such as blood or cell culture supernatants.

The present invention provides new materials and methods foridentifying, isolating, and purifying factor VIII and factor VIII-likeproteins, including REFACTO®, from a solution that contains suchproteins, in an active form. The factor VIII binding molecules of thepresent invention exhibit high affinity for factor VIII and factorVIII-like peptides. The current invention thus provides a cost-effectivemeans for rapid purification of commercial quantities of proteins usefulin the treatment of hemophilia A.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to provide novelbinding molecules for factor VIII and factor VIII-like proteins.Preferred binding molecules of the present invention exhibit not onlydistinct characteristics for binding of the target factor VIIIpolypeptides but also specific and desirable characteristics for release(elution) of the target polypeptides. Especially preferred bindingmolecules according to the invention are short polypeptide sequences,characterized by a stable loop structure.

A preferred method is disclosed herein for isolation of bindingmolecules according to the invention by employing phage displaytechnology. The phage display method of the current invention is usefulfor identifying families of polypeptide binding molecules, and usingthis technique several binding peptides exhibiting high affinity forfactor VIII and factor VIII-like peptides have been identified andisolated. Such binding peptides are useful for identifying, isolatingand purifying factor VIII and factor VIII-like polypeptides from asolution.

The most preferred binding molecules specific for factor VIII and factorVIII-like peptides isolated by the phage display method of the presentinvention are polypeptides characterized by a loop structure formed as aresult of a disulfide bond between two cysteine residues located at thepositions disclosed in I, II and III. Specific polypeptide bindingmolecules according to the present invention include polypeptidescomprising amino acid sequences of the following general formulas:X₁-X₂-Cys-X₃-X₄-X₅-X₆-X₇-Cys-X₈-X₉ (SEQ ID NO:1),  I.wherein X₁ is Arg, Phe, His or Pro; X₂ is Ser, Gly, Leu or His; X₃ isGly, Asn, Ile or Ser; X₄ is Ser, Trp or Gly; X₅ is Trp, Ile, Leu or Val;X₆ is Phe, Trp or Ser; X₇ is Pro or Phe; X₈ is Ser, Leu, Pro or Phe; X₉is Ala, Phe, Leu or His;X₁₀-X₁₁-Cys-X₁₂-X₁₃-Trp-X₁₄-X₁₅-Pro-Cys-X₁₆-X₁₇ (SEQ ID NO: 2),  II.wherein X₁₀ is Arg or His; X₁₁ is Ala, Mg, Gly, Leu or Pro; X₁₂ is Glyor Phe; X₁₃ is Ala or Ser; X₁₄ is Leu or Phe; X₁₅ is Mg, Asn or His; X₁₆is Ala, Asp, His, Leu, Phe, Pro, or Tyr; X₁₇ is Ala, Mg, Asn, Asp, orHis; andPhe-Cys-X₁₈-Val-X₁₉-X₂₀-Phe-X₂₁-His-Cys-X₂₂ (SEQ ID NO: 3),  III.wherein X₁₈ is His or Trp; X₁₉ is His or Phe; X₂₀ is Ala, Asn, His, orPro; X₂₁ is Ala, Asn, Asp, Gln, His, Leu, Ser, or Val; X₂₂ is Ala, Asp,His, Leu, Phe, or Ser.

In addition, it is also envisioned that the phage display method of thecurrent invention can also be used to isolate additional families ofbinding molecules specific for factor VIII and factor VII-likepolypeptides.

The most preferred binding molecules for isolation and/or purificationof factor VIII and factor VIII-like polypeptides, including especiallyREFACTO®, mentioned above, from a solution include the followingpolypeptides:

His-Ser-Cys-Gly-Ser-Trp-Leu-Phe- (SEQ ID NO: 4) Pro-Cys-Phe-Ala;Phe-Gly-Cys-Ser-Trp-Leu-Phe-Pro- (SEQ ID NO: 5) Cys-Pro-Phe;Pro-His-Cys-Asn-Trp-Leu-Phe-Pro- (SEQ ID NO: 6) Cys-Ser-Leu;Arg-Leu-Cys-Ser-Trp-Ile-Ser-Pro- (SEQ ID NO: 7) Cys-Ser-Ala;Phe-His-Cys-Ile-Gly-Val-Trp-Phe- (SEQ ID NO: 8) Cys-Leu-His;Arg-Leu-Cys-Ser-Trp-Val-Ser-Pro- (SEQ ID NO: 9) Cys-Ser-Ala;His-Pro-Cys-Gly-Ser-Trp-Leu-Arg- (SEQ ID NO: 10) Pro-Cys-Leu-His;Arg-Gly-Cys-Gly-Ser-Trp-Leu-Arg- (SEQ ID NO: 11) Pro-Cys-Leu-Asp;His-Pro-Cys-Gly-Ser-Trp-Leu-His- (SEQ ID NO: 12) Pro-Cys-Ala-Ala;His-Pro-Cys-Gly-Ser-Trp-Phe-Asn- (SEQ ID NO: 13) Pro-Cys-Ala-His;His-Pro-Cys-Gly-Ser-Trp-Phe-Arg- (SEQ ID NO: 14) Pro-Cys-Phe-His;His-Ala-Cys-Gly-Ser-Trp-Phe-Arg- (SEQ ID NO: 15) Pro-Cys-His-Ala;His-Leu-Cys-Gly-Ala-Trp-Phe-Arg- (SEQ ID NO: 16) Pro-Cys-Asp-Ala;His-Leu-Cys-Phe-Ala-Trp-Phe-Arg- (SEQ ID NO: 17) Pro-Cys-Asp-Ala;His-Gly-Cys-Gly-Ala-Trp-Phe-Arg- (SEQ ID NO: 18) Pro-Cys-His-Ala;His-Pro-Cys-Gly-Ala-Trp-Phe-Asn- (SEQ ID NO: 19) Pro-Cys-Pro-Arg;His-Pro-Cys-Gly-Ala-Trp-Leu-Arg- (SEQ ID NO: 20) Pro-Cys-Tyr-Asn;His-Arg-Cys-Gly-Ser-Trp-Leu-His- (SEQ ID NO: 21) Pro-Cys-Leu-Ala;Phe-Cys-Trp-Val-Phe-Ala-Phe-Asp- (SEQ ID NO: 22) His-Cys-His;Phe-Cys-Trp-Val-His-Pro-Phe-Ala- (SEQ ID NO: 23) His-Cys-Leu;Phe-Cys-His-Val-Phe-His-Phe-Ser- (SEQ ID NO: 24) His-Cys-Asp;Phe-Cys-Trp-Val-Phe-Ala-Phe-Asp- (SEQ ID NO: 25) His-Cys-His;Phe-Cys-Trp-Val-Phe-Asn-Phe-Ser- (SEQ ID NO: 26) His-Cys-Ser;Phe-Cys-Trp-Val-Phe-Pro-Phe-Asn- (SEQ ID NO: 27) His-Cys-Asp;Phe-Cys-Trp-Val-Phe-Pro-Phe-Asn- (SEQ ID NO: 28) His-Cys-Ser;Phe-Cys-Trp-Val-Phe-Pro-Phe-Gln- (SEQ ID NO: 29) His-Cys-Ala;Phe-Cys-Trp-Val-Phe-Pro-Phe-His- (SEQ ID NO: 30) His-Cys-Phe;Phe-Cys-His-Val-Phe-Asn-Phe-Val- (SEQ ID NO: 31) His-Cys-Ser;Phe-Cys-His-Val-Phe-Pro-Phe-Leu- (SEQ ID NO: 32) His-Cys-Asp;

Solutions from which factor VIII and factor VIII-like polypeptides canbe isolated and purified from include, but are not limited to, blood,blood fractions, and recombinant cell culture supernatants containingfactor VIII or a factor VIII-like polypeptide produced and secreted bythe recombinant host cell.

In another embodiment, the present invention provides a method foridentifying and isolating factor VIII binding molecules via phagedisplay technology. More specifically, the factor VIII and factorVIII-like binding molecules having specific and predetermined bindingand elution characteristics can be selected from a binding moleculelibrary, such as a phage display library, by a method comprising:

(a) selecting a first solution condition (i.e., the binding conditions)at which it is desired that a binding molecule should exhibit anaffinity for factor VIII or a factor VIII-like polypeptide, forming anaffinity complex;

(b) selecting a second solution condition (i.e., the release conditions)at which it is desired that the binding molecule will dissociate fromthe factor VIII or factor VIII-like polypeptide, wherein the secondsolution condition is different in some respect (e.g., temperature, pH,solvent concentration, etc.) from the first solution condition;

(c) providing a library of analogues of a parental factor VIII bindingdomain, wherein each analogue differs from said parental binding domainby variation of the amino acid sequence at one or more amino acidpositions within the domain;

(d) contacting said library of analogues with factor VIII or a factorVIII-like polypeptide at the first solution condition under conditionssuitable to form a complex between the binding molecule and a factorVIII or factor VIII-like polypeptide;

(e) removing from the solution the unbound members (analogues) of thebinding domain library;

(f) subjecting the factor VIII or factor VIII-like polypeptide complexesthat remain from step (e) to the second solution condition fordissociation of some of the binding molecule/factor VIII (or factorVIII-like polypeptide) complexes;

(g) recovering the binding analogues released under the second solutioncondition, wherein the recovered analogues identify isolated factor VIIIor factor VIII-like binding molecules.

Optionally, the above procedure can include additional release conditionsteps, i.e., optionally subjecting the factor VIII or factor VIII-likepolypeptide complexes that remain from step (f) to a third solutioncondition to dissociate other remaining complexes, which can becollected in a fraction separate from the factor VIII binding moleculesreleased under the second solution conditions. Such a step, if theconditions are stringent enough to dissociate all of the complexesformed in step (d), will identify solution conditions suitable forregeneration of binding matrices utilizing the binding moleculesisolated according to this process.

Also included in the present invention are non-peptide binding moleculesand modified polypeptides that bind factor VIII and/or factor VIII-likepolypeptides. An example of these modifications is a constrained-looppeptide having paired cysteine residues that form disulfide bonds,modified at the cysteine residues by substitution of one of thecysteines with non-natural amino acids capable of condensing with theother cysteine side-chain to form a stable thioether bridge. Such cyclicthioether analogues of synthetic peptides are described in PCTpublication WO 97/46251, incorporated herein by reference. Otherspecifically contemplated modifications include specific amino acidsubstitutions to lend stability or other properties withoutsignificantly affecting factor VIII binding, e.g., substitution ofGlu-Pro for Asp-Pro to reduce acid lability); N-terminal or C-terminalmodifications to incorporate linkers such as poly-glycine segments andalterations to include functional groups, notably hydrazide (—NH—NH₂)functionalities, e.g., to assist in immobilization of bindingpolypeptides according to this invention on solid supports.

In a further embodiment, the present invention encompasses a compositionof matter comprising isolated nucleic acids, preferably DNA, encodingbinding molecules of the present invention.

In another embodiment, the present invention provides a method fordetecting a factor VIII or a factor VIII-like peptide in a solutionsuspected of containing it, comprising contacting the solution with abinding molecule according to the invention and determining whether abinding complex has formed.

A further embodiment of the present invention is a method forpurification of factor VIII or a factor VIII-like polypeptide from asolution containing it, comprising the steps:

-   -   (a) contacting a solution containing factor VIII or a factor        VIII-like polypeptide with a binding molecule according to this        invention under solution conditions conducive to forming a        binding complex comprised of factor VIII or a factor VIII-like        polypeptide and the binding molecule;    -   (b) separating the complexes from the non-binding components of        the solution;    -   (c) dissociating the factor VIII or factor VIII-like polypeptide        from the binding molecule; and    -   (d) collecting the dissociated, purified factor VIII or factor        VIII-like polypeptide.

Also envisioned by the present invention is a method for isolatingfactor VIII and factor VIII-like peptides comprising:

-   -   (a) immobilizing a binding molecule according to the invention        on a solid support,    -   (b) contacting a factor VIII-containing solution or factor        VIII-like polypeptide-containing solution with the solid        support,    -   (c) removing the non-binding components from the solution, and    -   (d) eluting the factor VIII or factor VIII-like polypeptide from        the solid support.

One embodiment of the present invention pertains to polypeptides thatbind factor VIII. Specific polypeptide binding molecules according tothe present invention include polypeptides comprising amino acidsequences of the following:

Ser-Trp-X₁-X₂-Pro-Cys (SEQ ID NO: 45), wherein X₁ is Val, Ile, Leu orPhe, and wherein X₂ can be any amino acid;

X₁-X₂-Cys-Ser-Trp-X₃-X₄-Pro-Cys-X₅-X₆ (SEQ ID NO: 55), wherein: X₁ isArg or Phe; X₂ is Leu or Gly; X₃ is Val, Ile, Leu or Phe; X₄ is Ser orPhe; X₅ is Ser or Pro; and X₆ is Ala or Phe;

His-X₁-Cys-X₂-X₃-Trp-X₄-X₅-Pro-Cys-X₆-X₇ (SEQ ID NO: 59), wherein: X₁ isVal, Ile, Leu or Phe; X₂ is Gly or Phe; X₃ is Ala or Ser; X₄ is Leu orPhe; X₅ is Arg, Phe, Asn or His; X₆ is Tyr, Lys, Phe, Ala, Asp or His;and X₇ is Asn, His or Ala; and

Phe-Cys-X₁-Val-Phe-X₂-X₃-X₄-His-Cys-X₅ (SEQ ID NO: 70), wherein: X₁ isTrp or His; X₂ is Ala, Pro, Asn or Gin; X₃ is Phe or Trp; X₄ is Asp,Gln, Ser, Asn, Val, Arg or His; and X₅ is His, Ala, Ser, Asp or Phe.

Preferred binding molecules for isolation and/or purification of factorVIII and factor VIII-like polypeptides, from a solution include thefollowing polypeptides:

Ser-Trp-Val-Ser-Pro-Cys; (SEQ ID NO: 46) Ser-Trp-Leu-Phe-Pro-Cys; (SEQID NO: 47) Ser-Trp-Ile-Ser-Pro-Cys; (SEQ ID NO: 48)Ser-Trp-Leu-Arg-Pro-Cys; (SEQ ID NO: 49) Ser-Trp-Phe-Arg-Pro-Cys; (SEQID NO: 50) Ser-Trp-Leu-Phe-Pro-Cys; (SEQ ID NO: 51)Ser-Trp-Phe-Asn-Pro-Cys; (SEQ ID NO: 52) Ser-Trp-Leu-His-Pro-Cys; (SEQID NO: 53) Ser-Trp-Phe-Arg-Pro-Cys; (SEQ ID NO: 54)Arg-Leu-Cys-Ser-Trp-Val-Ser-Pro- (SEQ ID NO: 56) Cys-Ser-Ala;Phe-Gly-Cys-Ser-Trp-Leu-Phe-Pro- (SEQ ID NO: 57) Cys-Pro-Phe;Arg-Leu-Cys-Ser-Trp-Ile-Ser-Pro- (SEQ ID NO: 58) Cys-Ser-Ala;His-Pro-Cys-Gly-Ala-Trp-Leu-Arg- (SEQ ID NO: 60) Pro-Cys-Tyr-Asn;His-Pro-Cys-Gly-Ser-Trp-Leu-Arg- (SEQ ID NO: 61) Pro-Cys-Leu-His;His-Pro-Cys-Gly-Ser-Trp-Phe-Arg- (SEQ ID NO: 62) Pro-Cys-Phe-His;His-Ser-Cys-Gly-Ser-Trp-Leu-Phe- (SEQ ID NO: 63) Pro-Cys-Phe-Ala;His-Pro-Cys-Gly-Ser-Trp-Phe-Asn- (SEQ ID NO: 64) Pro-Cys-Ala-His;His-Leu-Cys-Phe-Ala-Trp-Phe-Arg- (SEQ ID NO: 65) Pro-Cys-Asp-Ala;His-Pro-Cys-Gly-Ser-Trp-Leu-His- (SEQ ID NO: 66) Pro-Cys-Ala-Ala;His-Ala-Cys-Gly-Ser-Trp-Phe-Arg- (SEQ ID NO: 67) Pro-Cys-His-Ala;His-Leu-Cys-Gly-Ala-Trp-Phe-Arg- (SEQ ID NO: 68) Pro-Cys-Asp-Ala; andHis-Arg-Cys-Gly-Ser-Trp-Leu-His- (SEQ ID NO: 69) Pro-Cys-Leu-Ala.

Particularly preferred binding molecules for isolation and/orpurification of factor VIII and factor VIII-like polypeptides from asolution include the following polypeptides:

Ser-Trp-Leu-His-Pro-Cys (SEQ ID NO: 53);

X₁-X₂-Cys-Ser-Trp-X₃-X₄-Pro-Cys-X₅-X₆ (SEQ ID NO: 55), wherein X₁ isArg, X₂ is Leu, X₄ is Ser, X₅ is Ser and X₆ is Ala;

His-X₁-Cys-X₂-X₃-Trp-X₄-X₅-Pro-Cys-X₆-X₇ (SEQ ID NO: 59), wherein X₁ isPro, X₂ is Gly, X₃ is Ser and X₅ is Arg;

His-Arg-Cys-Gly-Ser-Trp-Leu-His-Pro-Cys-Leu-Ala (SEQ ID NO: 69);

Phe-Cys-X₁-Val-Phe-X₂-X₃-X₄-His-Cys-X₅ (SEQ ID NO: 70), wherein X₁ isTrp and X₃ is Phe.

In another embodiment of the invention, the carboxyl and amino terminalends of the polypeptide comprise chemical modifications. In yet anotherembodiment, the chemical modification comprises a hydrazide functionalgroup a the carboxyl end and an acetylation at the amino end.

In another embodiment, the present invention pertains to a method forpurifying human factor VIII from a solution comprising: (a) immobilizinga factor VIII binding polypeptide of the invention, wherein thepolypeptide is attached to a solid support; (b) contacting a solutioncontaining human factor VIII with the polypeptide under conditions suchthat factor VIII binds to the polypeptide; and (c) separating boundfactor VIII from the solution, thereby purifying human factor VIII fromthe solution.

In another embodiment, the invention pertains to a compositioncomprising a polypeptide of the invention attached to a solid supportmatrix. In a preferred embodiment of the invention, the polypeptide isattached to the solid support via a covalent or non-covalent linkage.The solid support matrix is selected from the group consisting of:sepharose, agarose and cellulose.

In another embodiment, the present invention is directed to recombinantbacteriophage expressing exogenous DNA encoding one or more polypeptidesof the invention.

DEFINITIONS

As used herein, the term “recombinant” is used to describe non-naturallyaltered or manipulated nucleic acids, host cells transfected withexogenous nucleic acids, or polypeptides expressed non-naturally,through manipulation of isolated DNA and transformation of host cells.Recombinant is a term that specifically encompasses DNA molecules whichhave been constructed in vitro using genetic engineering techniques, anduse of the term “recombinant” as an adjective to describe a molecule,construct, vector, cell, polypeptide or polynucleotide specificallyexcludes naturally occurring such molecules, constructs, vectors, cells,polypeptides or polynucleotides.

The term “bacteriophage” is defined as a bacterial virus containing aDNA core and a protective shell built up by the aggregation of a numberof different protein molecules. The terms “bacteriophage” and “phage”are used herein interchangeably.

The term “factor VIII-like polypeptide” is used to refer to a modifiedor truncated form of natural factor VIII or full-length recombinantfactor VIII, which factor VIII-like polypeptide retains the procoagulantproperties of factor VIII. Examples of factor VIII-like polypeptides arethose active factor VIII fragments and factor VIII derivatives disclosedin the Andersson et al., Toole, and Almstedt et al. patents cited above,all of which are incorporated herein by reference. The term “factor VIIItarget” is sometimes used below to refer collectively to factor VIIIand/or factor VIII-like polypeptides contained in a solution orproduction feed stream.

The term “binding molecule” as used herein refers to any molecule,polypeptide, peptidomimetic or transformed cell (“transformant”) capableof forming a binding complex with another molecule, polypeptide,peptidomimetic or transformant. A “factor VIII binding molecule” is abinding molecule that forms a complex with factor VIII. In addition togeneral formulas, specific examples of factor VIII binding molecules arethe polypeptides described herein (e.g., SEQ ID NOS: 4-32, 36-44, 46-54,56-58, 60-69 and 71-79) and bacteriophage displaying any of suchpolypeptides. Also included within the definition of factor VIII bindingmolecules are polypeptides derived from or including a polypeptidehaving an amino acid sequence according to formula I, II or III, above,and such polypeptides which have been modified for particular results.Specific examples of modifications contemplated are C-terminal orN-terminal amino acid substitutions or polypeptide chain elongations forthe purpose of linking the binding moiety to a chromatographic supportor other substrate, and substitutions of pairs of cysteine residues thatnormally form disulfide links, for example with non-naturally occurringamino acid residues having reactive side chains, for the purpose offorming a more stable bond between those amino acid positions than theformer disulfide bond. All such modified binding molecules are alsoconsidered binding molecules according to this invention so long as theyretain the ability to bind factor VIII and/or factor VIII-likepolypeptides.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention makes possible the highly selective detection orpurification of factor VIII and/or factor VIII-like polypeptides in orfrom solutions containing them.

The factor VIII and factor VIII-like peptides can be produced in anyknown way, including chemical synthesis; production in transformed hostcells; secretion into culture medium by naturally occurring cells orrecombinantly transformed bacteria, yeasts, fungi, insect cells, andmammalian cells; secretion from genetically engineered organisms (e.g.,transgenic mammals); or in biological fluids or tissues such as blood,plasma, etc. The solution that contains the crude factor VIII as it isinitially produced (i.e., the production solution) will sometimes bereferred to as the “feed stream”.

Each method of producing factor VIII (or a factor VIII-like polypeptide)yields factor VIII in a feed stream that additionally contains a numberof impurities (with respect to the factor VIII). One purpose of thepresent invention is to produce affinity ligands and preparations (suchas chromatography media) comprising such ligands that allow rapid andhighly specific purification of factor VIII from a particular feedstream. The factor VIII affinity ligands obtained herein can be tailoredto the isolation of factor VIII from a particular feed stream, underspecific preselected conditions. If an alternate production method forthe factor VIII is used, producing a different feed stream, a differentset of affinity ligands may be necessary to achieve the same level ofpurification. The new set of ligands can be readily obtained followingthe procedures outlined herein.

Factor VIII binding molecules of the invention bind factor VIII withhigh affinity, comparable to or superior to other proteins such asantibodies known to bind factor VIII. Further, preferred affinityligands described herein release the factor VIII intact and in activeform under specific release conditions.

Selecting Binding and Release Conditions

Polypeptide binding molecules according to the present invention wereisolated using phage display technology, in a manner to identify factorVIII binding peptides exhibiting particular preselected properties ofbinding and release. According to this methodology, two solutionconditions can be preselected, i.e., binding conditions and releaseconditions. The binding conditions are a set of solution conditionsunder which it is desired that a discovered binding polypeptide willbind the target factor VIII (or factor VIII-like polypeptide); therelease conditions are a set of solution conditions under which it isdesired that a discovered binding polypeptide will not bind the factorVIII (i.e., will dissociate from factor VIII). The two conditions can beselected to satisfy any criterion of the practitioner, such as ease ofattaining the conditions, compatibility with other purification steps,lowered cost of switching between conditions compared to other affinitymedia, etc. Preferably, the two solution conditions are selected so asnot to adversely affect the stability or activity of the target protein(factor VIII or factor VIII-like polypeptide) and so as to differsignificantly with respect to at least one solution parameter. Forexample, in conducting the screening for suitable binding peptidesdescribed herein, binders were selected that dissociated from the targetin the presence of an ethylene glycol-containing buffer, or conditionsof lowered pH (i.e., pH 2), or combinations of those conditions, whichdiffered from the conditions employed for binding. Another parameterthat could be advantageously varied is the concentration of a salt, forexample NaCl, in the binding and elution buffers.

Selection of a Parental Binding Domain

In conjunction with selecting specific solution conditions for thedesired binding and release of the factor VIII, a parental bindingdomain is selected to serve as a structural template for the engineeredbinding molecules that will exhibit the desired binding and releasecapabilities. The binding domain can be a naturally occurring orsynthetic protein, or a region or domain of a protein. The parentalbinding domain can be selected based on knowledge of a known interactionbetween the parental binding domain and the factor VIII, but this is notcritical. In fact, it is not essential that the parental binding domainhave any affinity for factor VIII at all: Its purpose is to provide astructure from which a multiplicity of analogues (a “library”) can begenerated, which multiplicity of analogues will include one or moreanalogues that exhibit the desired binding and release properties (andany other properties selected for). The binding conditions and therelease conditions discussed supra can be selected with knowledge of theexact polypeptide that will serve as the parental binding domain, orwith knowledge of a class of proteins or domains to which the parentalbinding domain belongs, or completely independently of the choice of theparental binding domain. Similarly, the binding and/or releaseconditions can be selected with regard to known interactions between abinding domain and the factor VIII, e.g., to favor the interaction underone or both of the solution conditions, or they can be selected withoutregard to such known interactions. Likewise, the parental binding domaincan be selected taking into account the binding and/or releaseconditions or not, although it must be recognized that if the bindingdomain analogues are unstable under the binding or release conditions,no useful binding molecules may be obtained.

The nature of the parental binding domain greatly influences theproperties of the derived peptides (analogues) that will be testedagainst the factor VIII molecule. In selecting the parental bindingdomain, the most important consideration is how the analogue domainswill be presented to the factor VIII, i.e., in what conformation thefactor VIII and the analogues will come into contact. In preferredembodiments, for example, the analogues will be generated by insertionof synthetic DNA encoding the analogue into a replicable geneticpackage, resulting in display of the domain on the surface of amicroorganism, such as M13 phage, using techniques as described, e.g.,in Kay et al., Phage Display of Peptides and Proteins: A LaboratoryManual, (Academic Press, Inc.; San Diego 1996) and U.S. Pat. No.5,223,409 (Ladner et al.), incorporated herein by reference.

For formation of phage display libraries, it is preferred to usestructured polypeptides as the binding domain template, as opposed tounstructured, linear peptides. Mutation of surface residues in a proteinwill usually have little effect on the overall structure or generalproperties (such as size, stability, and temperature of denaturation) ofthe protein; while at the same time mutation of surface residues canprofoundly affect the binding properties of the protein. The moretightly a peptide segment is constrained, the less likely it is to bindto any particular target. If it does bind, however, the binding islikely to be tighter and more specific. Thus, it is preferred to selecta parental binding domain and, in turn, a structure for the polypeptideanalogues, that is constrained within a framework having some degree ofrigidity.

Preferably the protein domain that is used as the template or parentaldomain for generating the library of domain analogues will be a smallprotein or polypeptide. Small proteins or polypeptides offer severaladvantages over large proteins: First, the mass per binding site isreduced. Highly stable protein domains having low molecular weights,e.g., Kunitz domains (˜7 kDa), Kazal domains (˜7 kDa), Cucurbida maximatrypsin inhibitor (CMTI) domains (˜3.5 kDa), and endothelin (˜2 kDa),can show much higher binding per gram than do antibodies (150 kDa) orsingle-chain antibodies (30 kDa). Second, the possibility ofnon-specific binding is reduced because there is less surface available.Third, small proteins or polypeptides can be engineered to have uniquetethering sites in a way that is impracticable for larger proteins orantibodies. For example, small proteins can be engineered to havelysines only at sites suitable for tethering (e.g., to a chromatographymatrix), but this is not feasible for antibodies. Fourth, a constrainedpolypeptide structure is more likely to retain its functionality whentransferred with the structural domain intact from one framework toanother. For instance, the binding domain structure is likely to betransferable from the framework used for presentation in a library(e.g., displayed on a phage) to an isolated protein removed from thepresentation framework or immobilized on a chromatographic substrate.

Immobilization of the polypeptides according to the invention iscontemplated, e.g., onto chromatographic matrices to form efficientfactor VIII separation media for solutions such as whole blood orconditioned culture media containing factor VIII secreted from atransformant host cell. By selecting appropriate binding domaintemplates, binding polypeptides having a single free (unpaired withanother cysteine that ordinarily forms a disulfide link) cysteine can beisolated. Such thiol-functional polypeptides can be used for highlystable immobilization to substrates by formation of a thioether withiodoacetamide, iodoacetic acid, or similar α-iodo carboxylic acidgroups.

Similarly, the C-terminal carboxyl group of the polypeptide domain canbe converted to a hydrazide (—NH—NH₂), for reaction with analdehyde-functional substrate or other amine-reactive substrate. Thistechnique is preferred.

There are many small, stable protein domains suitable for use asparental binding domains and for which the following useful informationis available: (1) amino acid sequence, (2) sequences of severalhomologous domains, (3) 3-dimensional structure, and/or (4) stabilitydata over a range of pH, temperature, salinity, organic solvent, oxidantconcentration. Some examples are: Kunitz domains (58 amino acids, 3disulfide bonds), Cucurbida maxima trypsin inhibitor domains (31 aminoacids, 3 disulfide bonds), domains related to guanylin (14 amino acids,2 disulfide bonds), domains related to heat-stable enterotoxin IA fromgram negative bacteria (18 amino acids, 3 disulfide bonds), EGF domains(50 amino acids, 3 disulfide bonds), kringle domains (60 amino acids, 3disulfide bonds), fungal carbohydrate-binding domains (35 amino acids, 2disulfide bonds), endothelin domains (18 amino acids, 2 disulfidebonds), and Streptococcal G IgG-binding domain (35 amino acids, nodisulfide bonds). Most but not all of these contain disulfide bonds thatmaintain and stabilize the structure. The parental binding domain canalso be based on a single loop (one disulfide) of a microprotein that ishomologous to a known protein domain or not. For example, constrainedloops of 7 to 9 amino acids were used to form libraries for isolatingfactor VIII and factor VIII-like polypeptide binding molecules, asdescribed below. Libraries based on these domains, preferably displayedon phage, can be readily constructed and used for the selection ofbinding molecules according to this invention.

Providing a Library of Parental Binding Domain Analogues

Once a parental binding domain has been selected, a library of potentialbinding molecules is created for screening against a target, in thiscase factor VIII and/or factor VIII-like proteins, under the desiredbinding conditions and (optionally) the desired elution (release)conditions. The library is created by making a series of analogues, eachanalogue corresponding to the parental binding domain except having oneor more amino acid substitutions in the amino acid sequence of thedomain. The amino acid substitutions are expected to alter the bindingproperties of the domain without significantly altering its structure,at least for most substitutions. It is preferred that the amino acidpositions that are selected for variation (variable amino acidpositions) will be surface amino acid positions, that is, positions inthe amino acid sequence of the domains which, when the domain is in itsmost stable conformation, appear on the outer surface of the domain(i.e., the surface exposed to solution). Most preferably the amino acidpositions to be varied will be adjacent or close together, so as tomaximize the effect of substitutions. In addition, extra amino acids canbe added into the structure of the parental binding domain. In preferredembodiments, especially where a great deal of information is availableconcerning the interactions of factor VIII with other molecules,particularly the parental binding domain, those amino acid positionsthat are essential to binding interactions will be determined andconserved in the process of building the analogue library (i.e., theamino acids essential for binding will not be varied).

The object of creating the analogue library is to provide a very largenumber of potential binding molecules for reaction with the factor VIIImolecule, and in general the greater the number of analogues in thelibrary, the greater the likelihood that at least one member of thelibrary will bind to the factor VIII and release under preselected ordesirable conditions for release. Designed libraries following aparticular template structure and limiting amino acid variegation atparticular positions are much preferred, since a single library canencompass all the designed analogues and the included sequences will beknown and presented in roughly equal numbers. By contrast, randomsubstitution at only six positions in an amino acid sequence providesover 60 million analogues, which is a library size that begins topresent practical limitations even when utilizing screening techniquesas powerful as phage display. Libraries larger than this would poseproblems in handling, e.g., fermentation vessels would need to be ofextraordinary size, and more importantly, the likelihood of having allof the planned polypeptide sequence variations represented in theprepared library would decrease sharply. It is therefore preferred tocreate a designed or biased library, in which the amino acid positionsdesignated for variation are considered so as to maximize the effect ofsubstitution on the binding characteristics of the analogue, and theamino acid residues allowed or planned for use in substitutions arelimited, e.g., on the basis that they are likely to cause the analogueto bind under the solution conditions at which the library will bescreened for binders.

As indicated previously, the techniques discussed in Kay et al., supra,and Ladner et al., U.S. Pat. No. 5,223,409 are particularly useful inpreparing a library of analogues corresponding to a selected parentalbinding domain, which analogues will be presented in a form suitable forlarge-scale screening of large numbers of analogues with respect to atarget factor VIII molecule. The use of replicable genetic packages, andmost preferably bacteriophage, is a powerful method of generating novelpolypeptide binding entities that involves introducing a novel,exogenous DNA segment into the genome of a bacteriophage (or otheramplifiable genetic package) so that the polypeptide encoded by thenon-native DNA appears on the surface of the phage. When the insertedDNA contains sequence diversity, then each recipient phage displays onevariant of the template (parental) amino acid sequence encoded by theDNA, and the phage population (library) displays a vast number ofdifferent but related amino acid sequences.

In a screening procedure to obtain factor VIII binders according to thisinvention, a phage library is contacted with and allowed to bind atarget factor VIII molecule, usually immobilized on a solid support.Non-binders are separated from binders. In various ways, the bound phageare liberated from the factor VIII, collected and amplified. Since thephage can be amplified through infection of bacterial cells, even a fewbinding phage are sufficient to reveal the gene sequence that encodes abinding entity. Using these techniques it is possible to recover abinding phage that is about 1 in 20 million in the population. One ormore libraries, displaying 10-20 million or more potential bindingpolypeptides each, can be rapidly screened to find high-affinity factorVIII binders. When the selection process works, the diversity of thepopulation falls with each round until only good binders remain, i.e.,the process converges. Typically, a phage display library will containseveral closely related binders (10 to 50 binders out of 10 million).Indications of convergence include increased binding (measured by phagetiters) and recovery of closely related sequences. After a first set ofbinding peptides is identified, the sequence information can be used todesign other libraries biased for members having additional desiredproperties, e.g., discrimination between factor VIII and particularfragments or closely related impurities in a particular feed stream.

Such techniques make it possible not only to screen a large number ofpotential binding molecules but make it practical to repeat thebinding/elution cycles and to build secondary, biased libraries forscreening analog-displaying packages that meet initial criteria. Usingthese techniques, an analogue biased library can be screened to revealmembers that bind tightly (i.e., with high affinity) under the screeningconditions.

Synthesis of Polypeptide Analogues

Following the procedures outlined above, additional binding moleculesfor factor VIII and/or factor VIII-like polypeptides can be isolatedfrom the phage display libraries described herein or other phage displaylibraries or collections of potential binding molecules (e.g.,combinatorial libraries of organic compounds, random peptide libraries,etc.). Once isolated, the sequence of any individual binding peptide orthe structure of any binding molecule can be analyzed, and the bindercan be produced in any desired quantity using known methods. Forexample, the polypeptide binding molecules described herein, since theirsequences are now known, can advantageously be produced by chemicalsynthesis followed by treatment under oxidizing conditions appropriateto obtain the native conformation, i.e., the correct disulfide bondlinkages. Synthesis can be carried out by methodologies well known tothose skilled in the art (see, Kelley et al. in Genetic EngineeringPrinciples and Methods, (Setlow, J. K., ed.), Plenum Press, NY., (1990)vol. 12, pp. 1-19; Stewart et al., Solid-Phase Peptide Synthesis (1989),W. H. Freeman Co., San Francisco). The binding molecules of the presentinvention can be made either by chemical synthesis or by semisynthesis.The chemical synthesis or semisynthesis methods allow the possibility ofnon-natural amino acid residues to be incorporated.

During analysis of the specific polypeptides of the invention, threelibraries were isolated and screened. Table 10 shows the amino acidsequences of isolates from the libraries that scored highly on phageELISA tests. These libraries were designated TN7/1, TN8/6 anf TN9/1. Inaddition, important consensus sequences were determined.

The TN7/1 library isolates contain the 11 amino acid consensus sequenceX₁-X₂-Cys-Ser-Trp-X₃-X₄-Pro-Cys-X₅-X₆ (SEQ ID NO: 55), wherein X₁ is Argor Phe; X₂ is Leu or Gly; X₃ is Val, Ile, Leu or Phe; X₄ is Ser or Phe;X₅ is Ser or Pro; and X₆ is Ala or Phe. The following sequences wereidentified in the TN7/1 library as being factor VIII binders:

Arg-Leu-Cys-Ser-Trp-Val-Ser-Pro-Cys-Ser-Ala (SEQ ID NO: 56);

Phe-Gly-Cys-Ser-Trp-Leu-Phe-Pro-Cys-Pro-Phe (SEQ ID NO: 57); and

Arg-Leu-Cys-Ser-Trp-Ile-Ser-Pro-Cys-Ser-Ala (SEQ ID NO: 58).

The TN8/6 library isolates contain the 12 amino acid consensus sequenceHis-X₁-Cys-X₂-X₃-Trp-X₄-X₅-Pro-Cys-X₆-X₇ (SEQ ID NO: 59), wherein X₁ isVal, Ile, Leu or Phe; X₂ is Gly or Phe; X₃ is Ala or Ser; X₄ is Leu orPhe; X₅ is Arg, Phe, Asn or His; X₆ is Tyr, Lys, Phe, Ala, Asp or His;and X₇ is Asn, His or Ala. The following consensus sequences wereidentified in the TN8/6 library as being factor VIII binders:

His-Pro-Cys-Gly-Ala-Trp-Leu-Arg- (SEQ ID NO: 60) Pro-Cys-Tyr-Asn;His-Pro-Cys-Gly-Ser-Trp-Leu-Arg- (SEQ ID NO: 61) Pro-Cys-Leu-His;His-Pro-Cys-Gly-Ser-Trp-Phe-Arg- (SEQ ID NO: 62) Pro-Cys-Phe-His;His-Ser-Cys-Gly-Ser-Trp-Leu-Phe- (SEQ ID NO: 63) Pro-Cys-Phe-Ala;His-Pro-Cys-Gly-Ser-Trp-Phe-Asn- (SEQ ID NO: 64) Pro-Cys-Ala-His;His-Leu-Cys-Phe-Ala-Trp-Phe-Arg- (SEQ ID NO: 65) Pro-Cys-Asp-Ala;His-Pro-Cys-Gly-Ser-Trp-Leu-His- (SEQ ID NO: 66) Pro-Cys-Ala-Ala;His-Ala-Cys-Gly-Ser-Trp-Phe-Arg- (SEQ ID NO: 67) Pro-Cys-His-Ala;His-Leu-Cys-Gly-Ala-Trp-Phe-Arg- (SEQ ID NO: 68) Pro-Cys-Asp-Ala; andHis-Arg-Cys-Gly-Ser-Trp-Leu-His- (SEQ ID NO: 69) Pro-Cys-Leu-Ala.

The TN9/1 library isolates contain the 11 amino acid consensus sequencePhe-Cys-X₁-Val-Phe-X₂-X₃-X₄-His-Cys-X₅ (SEQ ID NO: 70), wherein X₁ isTrp or His; X₂ is Ala, Pro, Asn or Gln; X₃ is Phe or Trp; X₄ is Asp,Gln, Ser, Asn, Val, Arg or His; and X₅ is His, Ala, Ser, Asp or Phe. Thefollowing consensus sequences were identified in the TN9/1 library asbeing factor VIII binders:

Phe-Cys-Trp-Val-Phe-Ala-Phe-Asp- (SEQ ID NO: 71) His-Cys-His;Phe-Cys-Trp-Val-Phe-Pro-Phe-Gln- (SEQ ID NO: 72) His-Cys-Ala;Phe-Cys-Trp-Val-Phe-Asn-Phe-Ser- (SEQ ID NO: 73) His-Cys-Ser;Phe-Cys-Trp-Val-Phe-Pro-Phe-Asn- (SEQ ID NO: 74) His-Cys-Ser;Phe-Cys-Trp-Val-Phe-Asn-Trp-Val- (SEQ ID NO: 75) His-Cys-Asp;Phe-Cys-Trp-Val-Phe-Pro-Phe-Asn- (SEQ ID NO: 76) His-Cys-Asp;Phe-Cys-Trp-Val-Phe-Q-Phe-Arg-His- (SEQ ID NO: 77) Cys-His;Phe-Cys-His-Val-Phe-Asn-Phe-Val- (SEQ ID NO: 78) His-Cys-Ser; andPhe-Cys-Trp-Val-Phe-Pro-Phe-His- (SEQ ID NO: 79) His-Cys-Phe.

In one embodiment of the invention, the binding polypeptide comprisesthe consensus sequence Ser-Trp-X₁-X₂-Pro-Cys (SEQ ID NO: 45), wherein X₁is Val, Ile, Leu or Phe, and wherein X₂ can be any amino acid. In apreferred embodiment, the polypeptide comprises one or more of thefollowing amino acid sequences:

Ser-Trp-Val-Ser-Pro-Cys; (SEQ ID NO: 46) Ser-Trp-Leu-Phe-Pro-Cys; (SEQID NO: 47) Ser-Trp-Ile-Ser-Pro-Cys; (SEQ ID NO: 48)Ser-Trp-Leu-Arg-Pro-Cys; (SEQ ID NO: 49) Ser-Trp-Phe-Arg-Pro-Cys; (SEQID NO: 50) Ser-Trp-Leu-Phe-Pro-Cys; (SEQ ID NO: 51)Ser-Trp-Phe-Asn-Pro-Cys; (SEQ ID NO: 52) Ser-Trp-Leu-His-Pro-Cys; (SEQID NO: 53) and Ser-Trp-Phe-Arg-Pro-Cys. (SEQ ID NO: 54)In a particularly preferred embodiment, the consensus sequence isSer-Trp-Leu-His-Pro-Cys (SEQ ID NO: 53).

After a set of binding polypeptides is identified, the sequenceinformation can be used to design other secondary phage libraries,biased for members having additional desired properties. Once factorVIII binders have been initially isolated and characterized, furtherscreening for additional (“improved”) factor VIII binders can beperformed, for example, by creating a “biased” library derived from thediscovered consensus sequences. In one embodiment of the presentinvention, factor VIII binding polypeptides can be created throughsubstitution of specific amino acids. In a preferred embodiment,substitution occurs at amino acid positions that are not critical forbinding to factor VII or factor VII like polypeptides. These substationscan be performed to improve stability of the polypeptide, to add orremove proteolytic cleavage sites, add or remove additional bindingsites, or to increase or otherwise alter the length of the polypeptide.

Polypeptide binding molecules of the present invention are preferablyprepared using solid phase peptide synthesis (Merrifield, J. Am. Chem.Soc., 85: 2149 (1963); Houghten, Proc. Natl. Acad. Sci. USA, 82: 5132(1985)). Solid phase synthesis begins at the carboxy-terminus of theputative polypeptide by coupling a protected amino acid to a suitableresin, which reacts with the carboxy group of the C-terminal amino acidto form a bond that is readily cleaved later, such as a halomethylresin, e.g., chloromethyl resin and bromomethyl resin, hydroxymethylresin, aminomethyl resin, benzhydrylamine resin, ort-alkyloxycarbonyl-hydrazide resin. After removal of the α-aminoprotecting group with, for example, trifluoroacetic acid (TFA) inmethylene chloride and neutralizing in, for example, TEA, the next cyclein the synthesis is ready to proceed. The remaining α-amino and, ifnecessary, side-chain-protected amino acids are then coupledsequentially in the desired order by condensation to obtain anintermediate compound connected to the resin. Alternatively, some aminoacids can be coupled to one another forming an oligopeptide prior toaddition of the oligopeptide to the growing solid phase polypeptidechain.

The condensation between two amino acids, or an amino acid and apeptide, or a peptide and a peptide can be carried out according to theusual condensation methods such as azide method, mixed acid anhydridemethod, DCC (dicyclohexylcarbodiimide) method, active ester method(p-nitrophenyl ester method, BOP[benzotriazole-1-yl-oxy-tris(dimethylamino)phosphoniumhexafluorophosphate] method, N-hydroxysuccinic acid imido ester method),and Woodward reagent K method.

Common to chemical synthesis of peptides is the protection of thereactive side-chain groups of the various amino acid moieties withsuitable protecting groups at that site until the group is ultimatelyremoved after the chain has been completely assembled. Also common isthe protection of the α-amino group on an amino acid or a fragment whilethat entity reacts at the carboxyl group followed by the selectiveremoval of the α-amino-protecting group to allow subsequent reaction totake place at that location. Accordingly, it is common that, as a stepin the synthesis, an intermediate compound is produced which includeseach of the amino acid residues located in the desired sequence in thepolypeptide chain with various of these residues having side-chainprotecting groups. These protecting groups are then commonly removedsubstantially at the same time so as to produce the desired resultantproduct following purification.

The typical protective groups for protecting the α- and ε-amino sidechain groups are exemplified by benzyloxycarbonyl (Z),isonicotinyloxycarbonyl (iNOC), O-chlorobenzyloxycarbonyl [Z(NO₂)],p-methoxybenzyloxycarbonyl [Z(OMe)], t-butoxycarbonyl (Boc),t-amyloxycarbonyl (Aoc), isobomyloxycarbonyl, adamantyloxycarbonyl,2-(4-biphenyl)-2-propyloxycarbonyl (Bpoc), 9-fluorenylmethoxycarbonyl(Fmoc), methylsulfonylethoxycarbonyl (Msc), trifluoroacetyl, phthalyl,formyl, 2-nitrophenylsulphenyl (NPS), diphenylphosphinothioyl (Ppt),dimethylophosphinothioyl (Mpt), and the like.

As protective groups for the carboxy group there can be exemplified, forexample, benzyl ester (OBzl), cyclohexyl ester (Chx), 4-nitrobenzylester (ONb), t-butyl ester (Obut), 4-pyridylmethyl ester (OPic), and thelike. It desirable that specific amino acids such as arginine, cysteine,and serine possessing a functional group other than amino and carboxylgroups are protected by a suitable protective group as occasion demands.For example, the guanidino group in arginine can be protected withnitro, p-toluenesulfonyl, benzyloxycarbonyl, adamantyloxycarbonyl,p-methoxybenzenesulfonyl, 4-methoxy-2,6-dimethylbenzenesulfonyl (Mds),1,3,5-trimethylphenysulfonyl (Mts), and the like. The thiol group incysteine can be protected with p-methoxybenzyl, triphenylmethyl,acetylaminomethyl ethylcarbamoyl, 4-methylbenzyl, 2,4,6-trimethy-benzyl(Tmb), etc., and the hydroxyl group in the serine can be protected withbenzyl, t-butyl, acetyl, tetrahydropyranyl, etc.

After the desired amino acid sequence has been completed, theintermediate polypeptide is removed from the resin support by treatmentwith a reagent, such as liquid HF and one or more thio-containingscavengers, which not only cleaves the polypeptide from the resin, butalso cleaves all the remaining side-chain protecting groups. FollowingHF cleavage, the protein sequence is washed with ether, transferred to alarge volume of dilute acetic acid, and stirred at pH adjusted to about8.0 with ammonium hydroxide.

Upon pH adjustment, the polypeptide takes its desired conformationalarrangement.

Polypeptides according to the invention can also be preparedcommercially by companies providing polypeptide synthesis as a service(e.g., BACHEM Bioscience, Inc., King of Prussia, Pa.; Quality ControlledBiochemicals, Inc., Hopkinton, Mass.).

Use of the Binding Molecules in Detection or Purification

For detection of factor VIII and/or factor VIII-like polypeptides in asolution such as blood or conditioned media suspected of containing it,a binding molecule according to the invention can be detectably labeled,e.g., radiolabeled or enzymatically labeled, then contacted with thesolution, and thereafter formation of a complex between the bindingmolecule and the factor VIII target can be detected. A phage bindingmolecule according to the invention, i.e., a recombinant phagedisplaying a factor VIII binder polypeptide on its surface, can form acomplex with factor VIII and/or factor VIII-like polypeptides that isdetectable as a sediment in a reaction tube, which can be detectedvisually after settling or centrifugation.

Alternatively, a sandwich-type assay can be used, wherein a factor VIIIbinding molecule is immobilized on a solid support such as a plastictube or well, or a chromatographic matrix such as sepharose beads, thenthe solution suspected of containing the factor VIII target is contactedwith the immobilized binding molecule, non-binding materials are washedaway, and complexed factor VIII or factor VIII-like polypeptide isdetected using a suitable detection reagent, such as a monoclonalantibody recognizing the factor VIII target, which reagent is detectableby some conventional means known in the art, including being detectablylabeled, e.g., radiolabeled or labeled enzymatically, as withhorseradish peroxidase, and the like.

The binding molecules according to this invention will be extremelyuseful for isolation of factor VIII and/or factor VIII-like polypeptidesby affinity chromatography methods. Any conventional method ofchromatography can be employed. Preferably, an affinity ligand of theinvention will be immobilized on a solid support suitable, e.g., forpacking a chromatography column. The immobilized affinity ligand canthen be loaded or contacted with a feed stream under conditionsfavorable to formation of binding molecule/factor VIII (or factorVIII-like polypeptide) complexes. Non-binding materials can be washedaway, then the factor VIII (or factor VIII-like polypeptide) can beeluted by introducing solution conditions favoring dissociation of thebinding complex.

Alternatively, batch chromatography can be carried out by mixing asolution containing the factor VIII target and the binding molecule,then isolating complexes of the factor VIII target and the bindingmolecules. For this type of separation, many methods are known. Forexample, the binding molecule can be immobilized on a solid support,then separated from the feed stream along with the factor VIII target byfiltration. Or the binding molecule can be modified with its ownaffinity tag, such as a polyHis tail, which can be used to bind thebinder after complexes have formed using an immobilized metal affinitychromatography. Once separated, the factor VIII target can be releasedfrom the binding molecule under elution conditions and recovered in pureform.

It should be noted that although precise binding conditions werepreselected in obtaining the factor VIII-binding polypeptides disclosedherein, subsequent use in affinity purification can reveal more optimalbinding and release conditions under which the same isolated affinityligand will operate. Thus, it is not critical that the binding molecule,after isolation according to this invention, be always employed only atthe binding and release conditions that led to its separation from thelibrary.

Isolation of factor VIII binding molecules in accordance with thisinvention will be further illustrated below. The specific parametersincluded in the following examples are intended to illustrate thepractice of the invention, and they are not presented to in any waylimit the scope of the invention.

Example 1 The Isolation of Binding Molecules for a Factor VIII-LikePolypeptide

The techniques described above were employed to isolate high affinitybinding molecules for ligands for recombinantly produced factorVIII-like polypeptide consisting of two segments of human factor VIII,i.e., amino acids 1-743 and 1638 through 2332 of human factor VIII, asdescribed in U.S. Pat. No. 5,661,008 (Almstedt et al.), obtained underthe commercial designation of REFACTO® from Genetics Institute, Inc.(Cambridge, Mass.). The REFACTO® target was provided at a concentrationof about 530 μg/ml (7800 IU/ml) in 19.4 mM His, 300 mM NaCl, 3.4 mMCaCl₂ and 0.1% Tween 80, pH 7.0.

Three libraries, designated TN7 (5×10⁹ amino acid sequence diversity),TN8 (6×10⁹ amino acid sequence diversity), and TN9 (5×10⁹ amino acidsequence diversity), were constructed for expression of diversifiedpolypeptides on M13 phage. Each library was screened for binders topurified REFACTO®. Each of the libraries was constructed to display amicroprotein based on an 11- or 12-amino acid template. The TN7 libraryutilized a template sequence ofXaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa; the TN8 library utilized atemplate sequence of Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa;the TN9 library utilized a template sequence ofXaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Cys-Xaa.

Three rounds of screenings were carried out for each library. At theconclusion of the third round of screening eluted phage were propagated,and individual isolates from each library (96 per elution condition)were selected randomly and tested by standard ELISA techniques forbinding to the factor VIII target. Bound phage were detected with HRPconjugated anti-M13 polyclonal antibody (Pharmacia). TMB Peroxidasesubstrate was used for HRP in the ELISA detection mechanism. TMBsubstrate produces a blue color after peroxidase digestion. The color isquantitated by absorbance at OD₆₃₀. Phage isolates that provided asignificant signal (OD₆₃₀>0.25) above background were consideredpositive clones. DNA sequencing of these isolates was performed toidentify the displayed peptide.

Amino acid sequences of the displayed peptides were deduced from theobtained DNA sequences. Sequence data from the phage isolates weregrouped by library and sorted according to the degree of similarity. Thefrequency at which any given sequence was obtained was noted since thisindicates selection for a specific binder. Phage isolates having thesame display peptide were found to be present in phage populationsobtained by both of the two elution methods.

TABLE 1 Amino acid sequences of target-binding polypep- tides from theTN7 library SEQ TN7 frequency ELISA ID isolate sequence (elution) signalNO: A06 His-Ser-Cys-Gly-Ser- 7/96 0.5 4 Trp-Leu-Phe-Pro-Cys- (EG)Phe-Ala A08 Phe-Gly-Cys-Ser-Trp- 2/96 0.4 5 Leu-Phe-Pro-Cys-Pro- (EG)Phe D03 Pro-His-Cys-Asn-Trp- 7/192 0.2 6 Leu-Phe-Pro-Cys-Ser- (EG/pH2)Leu D04 Arg-Leu-Cys-Ser-Trp- 6/192 0.3 7 Ile-Ser-Pro-Cys-Ser- (EG/pH2)Ala A09 Phe-His-Cys-Ile-Gly- 2/192 0.1 8 Val-Trp-Phe-Cys-Leu- (EG/pH2)His C5/G10 Arg-Leu-Cys-Ser-Trp- 1/96 0.5 9 Val-Ser-Pro-Cys-Ser- (EG) Ala

TABLE 2 Amino acid sequences of target-binding polypep- tides from theTN8 library SEQ TN8 frequency ELISA ID isolate sequence (elution) signalNO: 10 His-Pro-Cys-Gly-Ser- 10/192 1.0 10 Trp-Leu-Arg-Pro-Cys- (EG/pH2)Leu-His 05 Arg-Gly-Cys-Gly-Ser-  2/192 0.2 11 Trp-Leu-Arg-Pro-Cys-(EG/pH2) Leu-Asp 04 His-Pro-Cys-Gly-Ser-  3/192 0.3 12Trp-Leu-His-Pro-Cys- (EG/pH2) Ala-Ala 02 His-Pro-Cys-Gly-Ser-  5/192 0.313 Trp-Phe-Asn-Pro-Cys- (EG/pH2) Ala-His 02 His-Pro-Cys-Gly-Ser-  3/960.7 14 Trp-Phe-Arg-Pro-Cys- (EG) Phe-His 07 His-Ala-Cys-Gly-Ser-  3/1920.4 15 Trp-Phe-Arg-Pro-Cys- His-Ala 02 His-Leu-Cys-Gly-Ala-  6/192 0.416 Trp-Phe-Arg-Pro-Cys- (EG/pH2) Asp-Ala 12 His-Leu-Cys-Phe-Ala-  1/960.4 17 Trp-Phe-Arg-Pro-Cys- (EG) Asp-Ala 01 His-Gly-Cys-Gly-Ala-  4/1920.2 18 Trp-Phe-Arg-Pro-Cys- (EG/pH2) His-Ala 01 His-Pro-Cys-Gly-Ala- 1/96 0.2 19 Trp-Phe-Asn-Pro-Cys- (pH2) Pro-Arg 08 His-Pro-Cys-Gly-Ala- 1/96 1.0 20 Trp-Leu-Arg-Pro-Cys- (EG) Tyr-Asn 11/G10His-Arg-Cys-Gly-Ser-  1/96 0.3 21 Trp-Leu-His-Pro-Cys- (EG) Leu-Ala

TABLE 3 Amino acid sequences of target-binding polypep- tides from theTN9 library SEQ N9 frequency ELISA ID isolate sequence (elution) signalNO: 04 Phe-Cys-Trp-Val-Phe-  6/192 0.8 22 Ala-Phe-Asp-His-Cys- (EG/pH2)His 02 Phe-Cys-Trp-Val-His-  2/96 0.2 23 Pro-Phe-Ala-His-Cys- (EG) Leu01 Phe-Cys-His-Val-Phe-  5/192 0.2 24 His-Phe-Ser-His-Cys- (EG/pH2) Asp01 Phe-Cys-Trp-Val-Phe- 12/192 1.2 25 Ala-Phe-Asp-His-Cys- (EG/pH2) His03 Phe-Cys-Trp-Val-Phe-  4/192 1.1 26 Asn-Phe-Ser-His-Cys- (EG/pH2) Ser02 Phe-Cys-Trp-Val-Phe-  5/96 0.4 27 Pro-Phe-Asn-His-Cys- (pH2) Asp 12Phe-Cys-Trp-Val-Phe-  6/96 1.0 28 Pro-Phe-Asn-His-Cys- (EG) Ser 09Phe-Cys-Trp-Val-Phe-  4/192 1.1 29 Pro-Phe-Gln-His-Cys- (EG/pH2) Ala 06Phe-Cys-Trp-Val-Phe-  2/192 0.3 30 Pro-Phe-His-His-Cys- (EG/pH2) Phe 01Phe-Cys-His-Val-Phe-  2/192 0.5 31 Asn-Phe-Val-His-Cys- (EG/pH2) Ser 11Phe-Cys-His-Val-Phe-  2/192 0.2 32 Pro-Phe-Leu-His-Cys- (EG/pH2) Asp

Example 2 Preparation of Affinity Ligands for a Factor VIII Target

Based on the data presented above, nine peptides were selected andsynthesized for immobilization on an affinity matrix material. Thepeptides synthesized are set forth in Table 4.

TABLE 4 Amino Acid Sequence of Affinity Ligands and their Densities onSolid Support Ligand Dens- ity Af- mg/ml finity Phage Sequence (μmol/Ligand Isolate (disulfide loop underlined) ml) CS-453 C10-TN8AEGTGDHPCGSWLRPCLHDPGPEGGGS- 2.64 NHNH₂ (0.98) CS-454 E02-TN8AEGTGDHLCGAWFRPCDADPGPEGGGS- 1.79 NHNH₂ (0.67) CS-455 A09-TN7AEGTGDFHCIGVWFCLHDPGPEGGGS- 2.21 NHNH₂ (0.83) CS-456 A08-TN7AEGTGDFGCSWLFPCPFDPGPEGGGS- 3.69 NHNH₂ (1.43) CS-458 B04-TN9AEGTGDFCWVFAFDHCHDPGPEGGGS- 3.15 NHNH₂ (1.17) CS-459 E09-TN9AEGTGDFCWVFPFQHCADPGPEGGGS- 2.72 NHNH₂ (1.02) CS-460 D06-TN9AEGTGDFCWVFPFHHCFDPGPEGGGS- 4.24 NHNH₂ (1.54) GI-1 C05/G10- Acetyl- 0.83TN7 AEGTGDRLCSWVSPCSADPEGGGSK (0.32) GI-2 A11/G10- Acetyl- 0.43 TN8AEGTGDHRCGSWLHPCLADPEGGGSK (0.16) The affinity peptides of Table 4 areidentified, in above order, with SEQ ID NOs: 36-44.

The nine lead affinity peptides were produced by classical solid-phasesynthetic methods as described above. To facilitate immobilization on asolid support, a short seven amino acid hydrazide-functional linkerregion (—PGPEGGGS—NHNH₂; SEQ ID NO: 34) was incorporated at thecarboxy-terminus of seven of the peptides (see Table 4). An alternativeimmobilization linker was used with two of the peptides (GI-1 and GI-2in Table 4), i.e., —PEGGGSK; (SEQ ID NO: 35), exhibiting a C-terminallysine for immobilization and an acetylated amino-terminus.

The candidate ligands were immobilized onto a formyl-substitutedethylene glycol-methacrylate chromatographic resin (Toyopearl Formyl650-M, pore size of ˜1000 Å; TosoHaas, Montgomeryville, Pa.). Thehydrazide-containing peptides were immobilized by facilitating hydrazonebond formation, the GI-1 and -2 peptides were immobilized via reductiveamination using NaCNBH₃. The amount of polypeptide immobilized on thesolid support was determined by quantifying the amount of freepolypeptide remaining in solution. The amount of ligand immobilized perml of resin was in the range of 0.7-1.5 μmol for thehydrazine-immobilized peptides.

The nine peptides were evaluated by affinity chromatography for theirability to capture the REFACTO® described in Example I, under specificbinding and release conditions. The buffers used in these evaluationsare set forth in Table 5.

TABLE 5 Binding and Elution Conditions Employed Binding Buffer 100 mMNH₄OAc, pH 6.3, 0.8 M NaCl, 1 M Sorbitol, 0.02% Tween 80, 3 mM EDTA, 5mM CaCl₂ Elution Buffer A 50% ethylene glycol, 20 mM His, 0.25 M NaCl,20 mM CaCl₂, 0.01% Tween 80, pH 7 Elution Buffer B 0.35 M CaCl₂, 20 mMHis, 0.3 M NaCl, 0.1% Tween 80, pH 7 pH 2 Clean 100 mM Gly, 1 M NaCl, pH2

The factor VIII-like polypeptide (REFACTO®) was diluted in SP Buffer toa concentration of 150 μg/ml. The affinity resins (˜350 μl) were eachpacked into glass columns, and approximately 150 μg of the factor VIIItarget was applied to the prepared affinity columns at a flow rate of200 μl/minute (linear velocity of 170 cm/hour). The bound material waseluted sequentially with the buffers as shown in Table 5, and proteinelution was monitored by UV absorbance at 280 nm. Fractions werecollected and the mass and activity of recovered factor VIII-likepolypeptide was determined by reversed-phase HPLC and by enzymaticassay.

For the mass determination, a standard curve with REFACTO® (0-200 μg)was generated and the amount present in each fraction was calculatedaccording to techniques well known in the art. Reversed-phase HPLC inthe presence of 20 mM EDTA was used to disrupt the REFACTO® moleculeinto its component subunits, which were eluted with a gradient ofacetonitrile/0.01% TFA. The activity assay was a Factor IX-, X-basedassay. The results for each affinity resin are set forth below (Table6).

TABLE 6 Summary of Data Obtained with Nine Affinity Ligands ElutionCondition (% recovery) Peptide Assay Flow A B pH2 Total UntreatedRP-HPLC 64.4 2.8 0 0 67.2 Resin Activity 64.4 0.6 65.0 CS-453 RP-HPLC 043.2 0 0 43.2 Activity 0 26.4 26.4 CS-454 RP-HPLC 2.5 45.1 0 0 47.6Activity 2.2 42.4 44.6 CS-455 RP-HPLC 65.8 1.4 0 0 67.2 Activity 61.61.3 62.9 CS-456 RP-HPLC 3.4 44.8 0 0 48.2 Activity 4.8 43.0 47.8 CS-458RP-HPLC 1.8 54.3 0 0 56.1 Activity 1.4 55.6 57.0 CS-459 RP-HIPLC 1.642.1 0 0 43.7 Activity 6.4 31.2 37.6 CS-460 RP-HPLC 24.6 28.8 0 0 53.4Activity 28.4 0 28.4 GI-1 RP-HPLC 65.7 0 0 0 65.7 Activity 64.0 0 64.0GI-2 RP-HPLC 31.3 28.1 0 2.0 61.4 Activity 33.7 20.3 53.9

In general, the total amount of the factor VIII target recovered afterchromatography over the nine ligands was in the range of 40-67%. Thepolypeptide ligands CS-453, CS-454, CS-456, and CS-459 capturedvirtually all of the factor VIII target applied, with bound materialbeing eluted in the presence of ethylene glycol. No activity was foundin the pH 2 eluant, therefore it was assumed that none of the targetremained bound to the ligand. The inability of the CS-455 and GI-1resins to capture the target can be due to degradation or instability ofthe peptide, or to low ligand density on the support.

Example 3 Comparative Binding of nhfVIII and REFACTO®

Experiments were conducted to demonstrate that the immobilizedpolypeptide ligands of Example II bind and release native human factorVIII (nhfVIII) under similar conditions and with similar yields asobserved with the factor VIII-like polypeptide REFACTO®.

For these experiments, nhfVIII was obtained from American Diagnostica,Inc. (Greenwich, Conn.; product #408 nat) in the form of a lyophilizedpowder containing stabilizing agents. The nhfVIII was reconstitutedaccording to the manufacturer's instructions in a reconstituting buffer(72 mM NH₄OAc, pH 6.3, 360 mM NaCl, 0.04% Tween 80 (Buffer 1).

A commercial ELISA kit (IMUBIND fVIII ELISA kit, Product #884, American,Inc., Greenwich, Conn.) developed to detect factor VIII was usedaccording to the manufacturer's specifications in order to detect boththe REFACTO® and the nhfVIII targets. The kit employs a sandwich ELISAassay in which the target is captured by an immobilized monoclonalantibody and the captured target is detected with a second monoclonalantibody-horseradish peroxidase (HRP) conjugate. Addition of theperoxidase substrate and its subsequent reaction with the HRP produces ablue color (detected at 630 nm) which changes to yellow (detected at 450nm) on addition of the 0.5N sulfuric acid stop solution. Color responseis calibrated with factor VIII standards provided by the manufacturer.

REFACTO® binding was tested in Buffer 1. The binding of both REFACTO®and nfhVIII were tested using three affinity resins prepared as inExample II, using the affinity peptides CS-454, CS-456, and CS-458immobilized on Toyopearl Formyl 650-M medium. Ligand density for eachpolypeptide was 1.79 mg/ml (0.67 μmol/ml), 3.69 mg/ml (1.43 μmol/ml) and3.15 mg/ml (1.17 μmol/ml) respectively.

For each of the three immobilized peptides tested, peptide-beads from200 ml of a 50% slurry of Toyopearl-coupled polypeptide suspension werecentrifuged briefly (30 seconds at 2000×g at room temperature), thesupernatant fluid was removed, and the beads (pellets) were washed twotimes. For each wash, the beads were resuspended in 500 μl of Buffer 1and centrifuged as before.

The stock solution of REFACTO® was diluted to a final concentration of200 U/ml in Buffer 1 and 250 μl of the diluted solution (˜50 U total)was added to a washed pellet of each of the peptide-beads. Thesuspension was incubated on an end-over-end mixer at RT for one hour,after which binding period the beads were pelleted by centrifugation (30seconds, 2000×g) and the supernatant solutions, representing the unboundfraction (“Unbound” in Table 7, below), were removed and retained forassay of unbound factor VIII activity.

The pelleted beads were washed one time by adding 250 μl of Buffer 1,mixed briefly and the suspension centrifuged as before. The supernatantsolutions (“Wash” in Table 7) were removed and retained for assay offactor VIII activity.

The washed pellets were resuspended in 250 μl of Buffer A (20 mML-Histidine-HCl, 250 mM NaCl, 20 mM CaCl₂, 0.01% Tween 80, 50% ethyleneglycol, pH 6.3) and incubated on an end-over-end mixer for 15 minutes atroom temperature. At the end of the elution period, the suspensions werecentrifuged as above. The supernatant solutions (“Eluate” in Table 7)were removed and retained for assay of eluted factor VIII activity.

The starting (diluted) REFACTO® solution (Input) and each sample(Unbound, Wash, and Eluate) taken as described above were diluted 1:1400in Assay Diluent (provided with kit), then subjected to ELISA using thecommercial factor VIII assay kit. Table 7 summarizes the results.

TABLE 7 Batch Binding and Elution of REFACTO ® with ImmobilizedPolypeptide Ligands Immobilized % of Input Recovered in: Peptide LigandInput Unbound Wash Eluate Total CS-454 100 24 12 49 85 CS-456 100 47 2024 91 CS-458 100 20 10 47 76

For each immobilized polypeptide tested, nearly all of the REFACTO®(>75%) added to the binding reaction was recovered in the Unbound, Wash,and Eluate fractions. A small amount of material (10%-25%) may have beenretained on the beads following elution.

Next the affinity beads were regenerated by one wash in 50% ethyleneglycol, 20 mM His, 0.25M NaCl, 20 mM CaCl₂, 0.01% Tween 80, pH 7, andtwo washes with 250 μl of 30 mM H₃PO₄, 1M NaCl, pH 2 (15 minutes foreach wash). Following the pH 2 washes, the beads were washed once in PBScontaining 0.05% azide and stored at 4° C.

A sample of nhfVIII was diluted to a final concentration of 100 U/ml byaddition of 2.32 ml H₂O, 180 μl 1M NH₄OAc, pH 6.3 (to 72 mM), and 1 μlTween 80 (to 0.04%). REFACTO® stock solution was diluted to 100 U/ml ina modified Buffer 1, in which the NaCl concentration was reduced from660 mM to 330 mM.

Immobilized peptides were tested for binding to nhFVIII in comparisonwith REFACTO®. As a non-binding control, a polypeptide from the TN9library (B10), which binds to an unrelated target and does not bind to afactor VIII target, was immobilized on the same methacrylate beads, asdescribed above. Next, nhfVIII and REFACTO® solutions were mixed withregenerated affinity beads bearing the CS-454, CS-456, and CS-458ligands in a comparative batch purification procedure. The reactionconditions are set forth in Table 8.

TABLE 8 Reaction Conditions for nhfVIII Binding Test Immobilized VolumeBead Slurry Target Reaction Volume Peptide Ligand (μl) (100 U/ml) (μl)CS-454 200 hfVIII 500 CS-456 200 hfVIII 500 CS-458 200 hfVIII 500TN9-B10 200 hfVIII 500 CS-458 100 REFACTO ® 250 TN9-B10 100 REFACTO ®250

The results of these trials are set forth in Table 9.

TABLE 9 Batch Binding and Elution of nhfVIII and REFACTO ® withImmobilized Polypeptide Ligands Immobilized % of Total Recovered in:Peptide Ligand Target Unbound Wash Eluate CS-454 nhfVIII 67 12 21 CS-456nhfVIII 70 14 16 CS-458 nhfVIII 48 13 39 TN9-B10 nhfVIII 86 14  0 CS-458REFACTO ® 59 14 27 TN9-B10 REFACTO ® 90 10  0

In conclusion, the immobilized polypeptide ligands, CS-458, CS-454, andCS-456 bind and release nhfVIII under similar conditions and withsimilar yields as observed previously with a factor VIII-likepolypeptide.

Example 4 Synthesis of Binding Polypeptides

One peptide was used for the purification factor VIII. This peptide isreferred to as “TN8.2”. The sequence of the peptide as it is synthesizedand used is:

Acetyl- AEGTGDHRCGSWLHPCLAEPGEGGGGSK. (SEQ ID NO: 33)

Binding polypeptides were synthesized with the amino terminal residueacetylated and the carboxyl terminal residue as a free acid. The twocysteine residues are oxidized and form a disulfide bond which creates acyclic peptide structure. The residues contained within the cyclicstructure are underlined. The peptide sequence is identical to thesequence GI-2 shown in Table 4 except that the aspartic acid residue atposition 19 has been changed to glutamic acid (bold). This change wasmade because the dipeptide sequence ASP PRO (DP) is not as stable asother dipeptide sequences and shows a relatively high rate ofhydrolysis, particularly at low pH. Changing the DP to EP improvedpeptide stability while retaining the general chemical properties of thesequence at that site.

Amino acid consensus sequences were discovered among peptide sequencesobtained from the selections. Table 10 shows the amino acid sequences ofisolates from the selections that scored highly on phage ELISA tests.Consensus sequences are present in these sequences. For example,Ser-Trp-X₁-X-Pro-Cys (SEQ ID NO: 45), which is present in the TN-7 andTN-8 library sequences; and Phe-Cys-Trp-Val-Phe-X-Phe-X-His-Cys-X (SEQID NO: 82), which is present in the TN-9 library sequences, wereidentified. The designation X₁ indicates the set of residues: VAL, ILE,LEU, or PHE, and X indicates that any amino acid can be present. Thesequences shown in Table 10 have highest affinity and specificity forthe target.

TABLE 10 High Scoring Isolates from Rounds 2 and 3 Li- bra- ry AAsequence N_(occ) ¹ R² Mode³ TN7/ RLCSWVSPCSA (SEQ ID NO: 56) 1 10 E 1FGCSWLFPCPF (SEQ ID NO: 57) 2 8 E RLCSWISPCSA (SEQ ID NO: 58) 4 6 E TN8/HPCGAWLRPCYN (SEQ ID NO: 60) 1 20 E 6 HPCGSWLRPCLH (SEQ ID NO: 61) 10 16E/2 HPCGSWFRPCFH (SEQ ID NO: 62) 3 16 E HSCGSWLFPCFA (SEQ ID NO: 63) 710 E HPCGSWFNPCAH (SEQ ID NO: 64) 4 8 E/2 HLCFAWFRPCDA (SEQ ID NO: 65) 18 2 HPCGSWLHPCAA (SEQ ID NO: 66) 1 6 2 HACGSWFRPCHA (SEQ ID NO: 67) 3 6E/2 HLCGAWFRPCDA (SEQ ID NO: 68) 6 6 E/2 HRCGSWLHPCLA (SEQ ID NO: 69) 16 E TN9/ FCWVFAFDHCH (SEQ ID NO: 71) 14 24 2 1 FCWVFPFQHCA (SEQ ID NO:72) 2 24 E FCWVFNFSHCS (SEQ ID NO: 73) 3 24 2 FCWVFPFNHCS (SEQ ID NO:74) 6 18 E FCWVFNWVHCD (SEQ ID NO: 75) 1 14 E FCWVFPFNHCD (SEQ ID NO:76) 6 8 2 FCWVFQFRHCH (SEQ ID NO: 77) 1 8 2 FCHVFNFVHCS (SEQ ID NO: 78)3 8 2 FCWVFPFHHCF (SEQ ID NO: 79) 1 6 E Notes: ¹N_(occ) is the number ofisolates that had the given sequence. ²R is the ratio of the ELISA scorewith target present to the ELISA score without target. ³Mode is the modeof elution used to obtain the given isolate. “E” means elution with 50%ethylene glycol, “2” means pH2 elution, and E/2 means peptide sequencefound in isolates obtained with both elution protocols.

TN7 r l C S W X₁ s P C S a (SEQ ID NO: 80) TN8 H p C G S W X₁ r P C X a(SEQ ID NO: 81) (φ = V, I, L, F) TN9 F C W V F X F X H C X (SEQ ID NO:82)

Following the foregoing description, the characteristics important foraffinity binding molecules permitting detection or separation of factorVIII or factor VIII-like polypeptides in or from any solution can beappreciated. Additional binding molecule embodiments of the inventionand alternative methods adapted to a particular solution or feed streamwill be evident from studying the foregoing description. All suchembodiments and obvious alternatives are intended to be within the scopeof this invention, as defined by the claims that follow.

Each of the publications referred to above is hereby incorporated byreference.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1. A polypeptide comprising the amino acid sequence X₁-X₂-Cys-Ser-Trp-X₃-X₄-Pro-Cys-X₅-X₆ (SEQ ID NO: 55), wherein: X₁ is Arg or Phe; X₂ is Leu or Gly; X₃ is Phe; X₄ is Ser or Phe; X₅ is Ser or Pro; and X₆ is Ala or Phe, such that the polypeptide binds factor VIII.
 2. The polypeptide of claim 1, wherein X₁ is Arg, X₂ is Leu, X₄ is Ser, X₅ is Ser and X₆ is Ala.
 3. The polypeptide of claim 1, wherein a terminal amino acid residue comprises a chemical modification.
 4. The polypeptide of claim 3, wherein the carboxyl terminus comprises a chemical modification.
 5. The polypeptide of claim 4, wherein the chemical modification comprises a hydrazide functional group.
 6. The polypeptide of claim 3, wherein the amino terminus has a chemical modification.
 7. The polypeptide of claim 6, wherein the chemical modification is an acetylation.
 8. A composition comprising a polypeptide comprising the amino acid sequence X₁-X₂-Cys-Ser-Trp-X₃-X₄-Pro-Cys-X₅-X₆ (SEQ ID NO: 55), wherein: X₁ is Arg or Phe; X₂ is Leu or Gly; X₃ is Phe; X₄ is Ser or Phe; X₅ is Ser or Pro; and X₆ is Ala or Phe, and wherein the polypeptide can bind factor VIII and the polypeptide is attached to a solid support matrix.
 9. The composition of claim 8 wherein the polypeptide is attached to the solid support matrix via a covalent or non-covalent linkage.
 10. The composition of claim 9, wherein the solid support matrix is selected from the group consisting of: sepharose, agarose and cellulose.
 11. A recombinant bacteriophage expressing exogenous DNA encoding a polypeptide, the polypeptide comprising the amino acid sequence X₁-X₂-Cys-Ser-Trp-X₃-X₄-Pro-Cys-X₅-X₆ (SEQ ID NO: 55), wherein: X₁ is Arg or Phe; X₂ is Leu or Gly; X₃ is Phe; X₄ is Ser or Phe; X₅ is Ser or Pro; and X₆ is Ala or Phe; such that the polypeptide binds factor VIII.
 12. The recombinant bacteriophage of claim 11, wherein X₁ is Arg, X₂ is Leu, X₄ is Ser, X₅ is Ser and X₆ is Ala. 